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1.
Neutron small angle scattering measurements of solutions of the Mo-Fe protein from C. pasteurianum have yielded the following results. The molecular weight of the protein is 208,000 ± 10,000, in agreement with figures obtained by other methods. The radius of gyration is 39.8 ± 0.7 Å in H2O, and 37.6 ± 0.3 Å in D2O. The experimental scattering curves have been compared with the calculated scattering curves of simple homogeneous bodies. It is concluded that the MoFe protein from C. pasteurianum is a non spherical particle having an axial ratio of 2:1, and that it probably has little, if any, solvent containing cavities.  相似文献   

2.
3.
The molecular weight and polypeptide chain stoichiometry of the native pyruvate dehydrogenase multienzyme complex from Escherichia coli were determined by independent techniques. The translational diffusion coefficient (Do20,w) of the complex was measured by laser light intensity fluctuation spectroscopy and found to be 0.90 (±0.02) × 10?11m2/s. When this was combined in the Svedberg equation with the measured sedimentation coefficient (so20,w = 60.2 (±0.4) S) and partial specific volume (v? = 0.735 (±0.01) ml/g), the molecular weight of the intact native complex was calculated to be 6.1 (±0.3) × 106. The polypeptide chain stoichiometry (pyruvate decarboxylase: lipoate acetyltransferase: lipoamide dehydrogenase) of the same sample of pyruvate dehydrogenase complex was measured by the radioamidination technique of Bates et al. (1975) and found to be 1.56:1.0:0.78.From this stoichiometry and the published polypeptide chain molecular weights estimated by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, a minimum chemical molecular weight of 283,000 was calculated. This structure must therefore be repeated approximately 22 times to make up the native complex, a number which is in good agreement with the expected repeat of 24 times if the lipoate acetyltransferase core component has octahedral symmetry. It is consistent with what appears in the electron microscope to be trimer-clustering of the lipoate acetyltransferase chains at the corners of a cube. It rules out any structure based on 16 lipoate acetyltransferase chains comprising the enzyme core.The preparation of pyruvate dehydrogenase complex was polydisperse: in addition to the major component, two minor components with sedimentation coefficients (so20,w) of 90.3 (±0.9) S and 19.8 (±0.3) S were observed. Together they comprised about 17% of the total protein in the enzyme sample. Both were in slowly reversible equilibrium with the major 60.2 S component but appeared to be enzymically active in the whole complex reaction. The faster-sedimenting species is probably a dimer of the complex, whereas the slower-sedimenting species has the properties of an incomplete aggregate of the component enzymes of the complex based on a trimer of the lipoate acetyltransferase chain.  相似文献   

4.
The molecular weight distribution of sinistrin (Inutest ®, Laevosan Ges., Linz, Austria), determined by analytical gel-permeation chromatography, using narrow fractions (MwMn< 1.07) obtained by preparative gel-permeation chromatography, covered the range 800–16,000 with Mn  2,500 and Mw  3,500. From viscosity measurements on dilute, aqueous solutions, the relation [η]  0.28 X M0.3 was obtained, indicating a branched molecular structure; the largest molecules can be described by a sphere with r  23 Å. Comparison of the content of glucose and reducing sugars in the fractions with the molecular weight determined by vapour-pressure osmometry indicated that a glucose end-group is present in the majority of the molecules. The percentage of glucose end-groups is higher in the fractions of lower molecular weight. From this finding, speculations on the biosynthesis of sinistrin are made. The specific optical rotation of sinistrin fractions decreases linearly with 1/Mn.  相似文献   

5.
An Fab fragment from a monoclonal antibody (NTS10/1) has been crystallized. The antigen, influenza virus A/Tokyo/3/67 (N2) neuraminidase, is also crystalline and its structure analysis is in progress. The Fab crystals are trigonal, space group P3121 with cell dimensions a = 132·3 A?, c = 73·8 A?. This crystalline material forms a complex with the neuraminidase with an equilibrium binding constant in excess of 1011m?1. The molecular weight of the complex is 406,000 ± 20,000 indicating that four Fab fragments are attached to each neuraminidase tetramer. The separate crystallization of antigen and Fab fragment opens the way to map, for the first time, the complementary surfaces of an antigen-antibody complex.  相似文献   

6.
Crystals of β-lactamase I from Bacillus cereus 569 are monoclinic, space group C2 with unit cell dimensions a = 143·0 (± 0·5), b = 35·8 (± 0·1), c = 52·7 (± 0·2) A?, β = 97·0 (± 0·1) °, and one molecule of molecular weight about 28,000 per asymmetric unit.  相似文献   

7.
A pencillin-sensitive enzyme, the exocellular dd-carboxypeptidase-transpeptidase from Streptomyces R61, has been crystallized from polyethylene glycol (Mr = 6000 to 7500) solution at pH 7·6. X-ray examination of the orthorhombic crystals shows the space group is P212121, with unit cell dimensions a = 51·1 A?, b = 67·4 A?, and c = 102·9 A?. With four molecules of molecular weight 38,000, the A?3/dalton ratio for the cell is 2·33. The crystals are stable to irradiation for 75 hours and are suitable for structure analysis to at least 2·4 Å resolution. The radius of gyration of the molecule in solution at pH 6.8 is 20.8 Å.  相似文献   

8.
Crystals of the bacteriochlorophyll-protein from the green photosynthetic bacterium Chlorobium limicola, previously described by J. M. Olson et al. (1969), have been re-examined by X-ray diffraction. The space group is P63, as reported in the earlier work, but revised cell dimensions, a = b = 112.4 ± 0.4 A?, c = 98.4 ± 0.4 A?, were obtained, leading to a unit cell volume one third of that reported previously. Correction of this error leads to the conclusion that the bacteriochlorophyll-protein complex must be a trimer consisting of three identical subunits arranged about a crystallographic symmetry axis. Also a new trigonal crystal form of the bacteriochlorophyll-protein has been obtained, and is consistent only with a molecule composed of three identical or near-identical subunits. Models of the molecular packing for both crystal forms are presented.The molecular weight of the bacteriochlorophyll-protein complex, determined from crystal density measurements, is (1.53 ± 0.23) × 105, and the overall molecular dimensions are about 55 Å along the trimer axis, and 83 Å at right angles to this. There are probably seven bacteriochlorophyll molecules in each subunit.  相似文献   

9.
Crystals of mitogenic lectin from pea seeds (Pisum sativum) have been grown. The crystals are in space group P212121 with unit cell dimensions: a = 51.0 ± 0.2 A?, b = 617 ± 0.2 A?, c = 137.6 ± 0.5 A?. The asymmetric unit has one protein molecule.  相似文献   

10.
Pseudomonas aeruginosa cytochrome oxidase (nitrite reductase, cytochrome cd) has been crystallized in space group P21212 with cell dimensions a = 122.8 A?, b = 87.2 A?, c = 73.4 A?. Density measurements suggest that the asymmetric unit contains one 63,000 molecular weight subunit of the dimeric molecule. Crystal data agree well with electron microscopy of single molecules. The X-ray pattern extends beyond 2.5 Å resolution, and structure analysis is in progress.  相似文献   

11.
Four subunits of the acetylcholine receptor molecule, obtained from the electric organ of Torpedo ocellata, have been isolated using polyacrylamide gel electrophoresis, and assayed by titration with a fluorescent lanthanide, terbium, and by affinity-labeling with p-(N-maleimido)benzyl [trimethyl-3H] ammonium iodide. The site with which the activator-analogue affinity label reacts, as well as the terbium-binding sites, are mainly associated with the smallest of the subunits of an apparent molecular weight of 40,000. Calcium competes with terbium for these binding sites. The affinity for terbium is the same in the intact molecule as in the subunit (KTb ? 19 ± 1 μM), but the affinity for calcium decreases by a factor of 4 (KCa ? 4 mM) in the subunit. Hydrolysis of the receptor, catalyzed by trypsin and chymotrypsin, to peptides with an apparent molecular weight of 8000 or less, does not affect the terbium-binding sites. These experiments indicate that the binding sites for neural activators and for calcium are associated with the same subunit, and that the terbium- and calcium-binding sites reflect structural properties of the polypeptide chain rather than the three-dimensional structure of the protein.  相似文献   

12.
Injections of 1 mg PGI2 directly into the bovine corpus luteum significantly increased peripheral plasma progesterone concentrations within 5 min. Concentrations were higher in the PGI2-treated heifers than in saline-injected controls between 5 and 150 min and at 3.5, 4, 5, and 7 h post-treatment. Levels tended to remain elevated through 14 h. Saline and 6-keto-PGF were without effect on plasma progesterone levels. The luteotrophic effect of PGI2 was not due to alterations in circulating LH concentrations. An in vitro experiment assessed the effects of either PGI2 alone or in combination with LH on progesterone production by dispersed luteal cells. Progesterone accumulation over 2 h for control, 5 ng LH, 1 μg PGI2, 10 μg PGI2, and 10 μg PGI2 plus 5 ng LH averaged 99 ± 42, 353 ± 70, 152 ± 35, 252 ± 45, and 287 ± 66 ng/ml (n=4), respectively. Thus PGI2 has luteotrophic effects on the bovine CL both in vivo and in vitro.  相似文献   

13.
In order to investigate the role of the plasma membrane in determining the kinetics of removal of cholesterol from cells, the efflux of [3H]cholesterol from intact cells and plasma membrane vesicles has been compared. The release of cholesterol from cultures of Fu5AH rat hepatoma and WIRL-3C rat liver cells to complexes of egg phosphatidylcholine (1 mg / ml) and human high-density apolipoprotein is first order with respect to concentration of cholesterol in the cells, with half-times (t12) for at least one-third of the cell cholesterol of 3.2 ± 0.6 and 14.3 ± 1.5 h, respectively. Plasma membrane vesicles (0.5–5.0 μm diameter) were produced from both cell lines by incubating the cells with 50 mM formaldehyde and 2 mM dithiothreitol for 90 min. The efflux of cholesterol from the isolated vesicles follows the same kinetics as the intact, parent cells: the t12 values for plasma membrane vesicles of Fu5AH and WIRL cells are 3.9 ± 0.5 and 11.2 ± 0.7 h, respectively. These t12 values reflect the rate-limiting step in the cholesterol efflux process, which is the desorption of cholesterol molecules from the plasma membrane into the extracellular aqueous phase. The fact that intact cells and isolated plasma membranes release cholesterol at the same rate indicates that variations in the plasma membrane structure account for differences in the kinetics of cholesterol release from different cell types. In order to investigate the role of plasma membrane lipids, the kinetics of cholesterol desorption from small unilamellar vesicles prepared from the total lipid isolated from plasma membrane vesicles of Fu5AH and WIRL cells were measured. Half-times of cholesterol release from plasma membrane lipid vesicles of Fu5AH and WIRL cells were the same, with values of 3.1 ± 0.1 and 2.9 ± 0.2 h, respectively. Since bilayers formed from isolated plasma membrane lipids do not reproduce the kinetics of cholesterol efflux observed with the intact plasma membranes, it is likely that the local domain structure, as influenced by membrane proteins, is responsible for the differences in t12 values for cholesterol efflux from these cell lines.  相似文献   

14.
(1) Alkyl sugar inhibition of d-allose uptake into adipocytes has been used to explore the spatial requirements of the external sugar transport site in insulin-treated cells. α-methyl and β-methyl glucosides show low affinity indicating very little space around C-1. The high affinity of d-glucosamine (Ki = 9.05 ± 0.66 mM) is lost by N-acetylation. N-Acetyl-d-glucosamine shows no detectable affinity, indicating that a bulky group at C-2 is not accepted. Similarly 2,3-di-O-methyl-d-glucose (Ki = 42.1 ± 7.5 mM) has lower affinity than 3-O-methyl-d-glucose (Ki = 5.14 ± 0.32 mM) indicating very little space around C-2 but much more around C-3. A reduction in affinity does occur if a propyl group is introduced into the C-3 position. The Ki for 3-O-propyl-d-glucose is 11.26 ± 2.12 mM. 6-O-Methyl-d-galactose (Ki = 87.2 ± 17.9 mM) and 6-O-propyl-d-glucose (Ki = 78.07 ± 12.6 mM) show low affinity compared with d-galactose and d-glucose, indicating steric constraints around C-6. High affinity is restored in 6-O-pentyl-d-galactose (Ki = 4.66 ± 0.23 mM) possibly indicating a hydrophobic binding site around C-6). (2) In insulin treated cells 4,6-O-ethylidene-d-glucose (Ki = 6.11 ± 0.5 mM) and maltose (Ki = 23.5 ± 2.1 mM) are well accommodated by the site but trehalose shows no detectable inhibition. These results indicate that the site requires a specific orientation of the sugar as it approaches the transporter from the external solution. C-1 faces the inside while C-4 faces the external solution. (3) To determine the spatial and hydrogen bonding requirements for basal cells 40 μM 3-O-methyl-d-glucose was used as the substrate. Poor hydrogen bonding analogues and analogues with sterically hindering alkyl groups showed similar Ki values to those determined for insulin-treated cells. These results indicate that insulin does not change the specificity of the adipocyte transport system.  相似文献   

15.
The micellar properties of gangliosides in water solutions were investigated by quasielastic light scattering measurements. GM1 and GD1a gangliosides were isolated from calf brain, purified to more than 99% and dissolved in 0.025 M Tris—HCI buffer (pH 6.8) at 37°C. The average intensity of scattered light and the intensity correlation function were measured by an apparatus including a 5145 Å argon laser and a real-time digital correlator. The scattered intensity data allowed the derivation of an upper limit to the critical micelle concentration (c0) and the evaluation of the molecular weight (M) of the micelle. The intensity correlation function gave the diffusion coefficient D, and hence the hydrodynamic radius RH, and also contained information on the polydispersity of the sample. We find co < 1 × 10?6 M for both GM1 and GD1a, M = 532 000 ± 50 000 and RH = 63.9 ± 2 A? for GM1, and M = 417 000 ± 40 000 and RH = 59.5 ± 2 A? for GD1a. The mixture 3:1 of the two gangliosides gave intermediate values for all examined parameters. The presence of cations, within the physiological concentration range. and, in particular of Ca2+, did not influence significantly the values of co and the main features of the micelle.  相似文献   

16.
Plasma membranes were isolated from HM7 melanoma cells grown in the presence of [3H]glucosamine and Na235SO4 or [3H]mannose and [14C]glucosamine. The labelled glucoconjugates were solubilized with 0.6 M lithium diiodosalicylate/0.5% Triton X-100. Fractionation of glycoconjugates by repeated chromatography on columns of Sepharose CL-6B and DEAE-Sepharose and by affinity chromatography on WGA-Sepharose yielded three radiochemically homogenous glycoproteins. One of these having an apparent molecular weight of 100 000 was found to contain clusters of (AcNeu)1 or in2 å [Gal å GalNAc] linked O-glycosidically to the protein. One other glycoprotein contained both O-glycosidically and N-glycosidically-linked oligosaccharides, and the third contained only N-glycosidically-linked carbohydrates. Preliminary results indicate that the 100 000 molecular weight mucin-type glycoprotein is present in significantly reduced quantities in cultured human fetal uveal melanocytes. Further, the bulk of the glycoproteins from the melanocytes were of lower molecular size compared to those from the melanoma cells.  相似文献   

17.
Large single crystals of isocitrate dehydrogenase from Azotobacter vinelandii have been grown by vapor diffusion from ammonium sulfate and phosphate solutions. The crystals are tetragonal, space group P42212 with cell dimensions a = 122.1 A?, c = 163.9 a?. There are two molecules of 80,000 molecular weight per asymmetric unit. Native data to 5.5 Å resolution have been collected on a diffractometer. A rotation function using data between 10 Å and 6 Å resolution indicates three possible orientations of the non-crystallographic 2-fold axis relating the two molecules.  相似文献   

18.
The insecticidal toxin of Bacillusthuringiensis subsp. kurstaki was isolated from parasporal crystals. The toxin, which is stable for several months, is a glycoprotein with an apparent molecular weight of 68,000 that is generated upon solubilization and activation of a higher molecular weight protoxin (MWapp = 1.3 × 105) at alkaline pH. The toxin was purified by gel filtation and anion exchange chromatography and its molecular weight was established by gel filtration chromatography and SDS polyacrylamide gel electrophoresis.  相似文献   

19.
Exposure of human erythrocyte ghosts (pH 8, 10°C) to visible light in the presence of the photosensitizer, methylene blue, results in a relatively rapid loss of spectrin (bands 1 and 2 on sodium dodecyl sulfate gel electropherograms) and the appearance of high molecular weight cross-linked derivatives. Isolated spectrin also undergoes photosensitized cross-linking, indicating that the reaction is not lipid-dependent.Extensive cross-linking was neither reversed by dithiothreitol nor prevented by prior blocking of SH groups with N-ethylmaleimide, suggesting that cysteine residues are not crucial bridging sites. The possible requirement for NH2 groups, as suggested by previous model studies (Dubbelman, T.M.A.R., de Goeij, A.F.P.M. and van Steveninck, J. (1978) Biochim. Biophys. Acta 511, 141–151), was tested. Succinylation of spectrin protected against cross-linking, but this effect is attributed to the disruption of quaternary structure, as deduced from sedimentation measurements. However, virtually complete blocking of NH2 groups by amidination perturbed overall structure relatively little, and had no effect on cross-linking. Moreover, exogenous amines such as ethylamine, added in large excess to spectrin prior to irradiation, did not interfere with cross-link formation. These results suggest that NH2 groups are not involved in the reaction.  相似文献   

20.
Cytochrome c7, a low potential, three heme-containing protein (molecular weight 9800) was isolated from Desulfuromonas acetoxidans and crystallized from polyethylene glycol solutions. The crystals belong to the orthorhombic space group P212121, with unit-cell dimensions a = 44·10 A?, b = 37·55 A? and c = 45·08 A? and have one molecule of protein per asymmetric unit.  相似文献   

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